The organizing principle behind this investigation is the close relationship between these two phosphohydrolases, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which occur together in relatively high concentrations in vertebrate intestinal mucosa. Presently available evidence suggests similarities in the structure and catalytic mechanism of these two enzymes. In contrast to the intensively-studied alkaline phosphatase, relatively little is known about the phosphodiesterase; this investigation will focus on elucidation of this enzyme's structure and function, which will be compared in detail to alkaline phosphatase. The major objective is the detection, isolation, and chemical and enzymic characterization of a covalant enzyme-substrate intermediate. This includes not only a chemical description of the intermediate, its mode and site of attachment to the enzyme (including amino acid sequence of an active site peptide), but also the effect of various alternate substrates, inhibitors, metal ions, chelators, and other potential modifiers on the amount of intermediate and the rate of its formation and discharge. Specifically included in these studies will be phosphonate esters, which we have found to be specific substrates for this phosphodiesterase, as well as several other untested compounds of potential interest. In the event that, despite the present positive indications, no covalent intermediate is found, an active site peptide will be labeled by alternative techniques and chemically characterized. The results obtained with 5'-nucleotide phosphodiesterase will be compared with the previously reported properties of alkaline phosphatase in the light of the hypothesis that these two enzymes share a common ancestor.